Isoprenoid biosynthesis is an important cellular metabolic pathway and is responsible for synthesis of structurally diverse and biologically active class of compounds called terpenoids, which includes sterols, steroidal sapogenins, alkaloids and lactones in plants. Withanolides is a group of naturally occurring steroidal lactones commonly present in members of Solanaceae. Majority of secondary metabolites (SMs) concentrated in Withania somnifera are responsible for defence, signalling, flavour, fragrance, hormonal, antibiotic, insecticidal, pharmacological and therapeutic efficiencies. Withania somnifera (L.) Dunal, commonly known as Indian ginseng, is principally recognized for its medicinal value in Ayurveda. Biological activity of withanolides, especially withanolide A and withaferin A have been studied extensively for their anti-cancerous properties (Jayaprakasam et al. 2003; Ichikawa et al. 2006). Plant extracts from different tissues are found effective in treatment of arthritis, geriatric problems, nervous and venereal disorders. Withanolides have been widely studied for their antioxidant, anti-inflammatory, immunomodulation, antiserotogenic, adaptogenic, rejuvenating effect and for protection against carbon tetrachloride induced hepatotoxicity. Some withanolides have been found associated with dendrite extension (Tohda et al. 2000) and inducing neurite outgrowth in human neuroblastoma SH-SY5Y cells (Zhao et al. 2002).
Withanolides are organic compounds characterized by C28 ergostane type steroid backbone and a side chain of C9 units of which a distinctive feature is the six-membered lactone ring which accounts for the plant biological efficacies. The basic skeleton of a withanolide is defined as a 22-hydroxyergostan-26-oic acid-26, 22-lactone and are classified on the basis of their structural variations derived by modifications either on the carbocyclic skeleton or side chain. Currently more than 40 withanolides and several sitoindosides (withanolides with glucose molecule at C-27) have been isolated from aerial parts, roots and berries of Withania species.
Withania somnifera is a slow growing shrub requiring dry conditions and produces minimal quantity of withanolides which are found to be localized mainly in leaves and roots with concentration ranging from 0.001-0.5% dry weight (Mirjalili et al. 2009). Such miniscule concentrations of withanolides are incapable of accomplishing the tremendously increasing economic demand for medicinal formulations.
Alternatively, chemical synthesis of withanolides have been attempted (Kovganko and Kashkan 1997; Gamoh et al. 1984; Jana et al. 2011), but it requires extensive experimentation due to the structural complexities and specific stereochemical requirements of the compounds resulting in low yield, hence the process is not feasible for secondary metabolite production.
Grover et al (J Biosci Bioeng. 2013 June; 115(6):680-5) reports enhanced withanolide production by overexpression of SQS in Withania somnifera using Agrobacterium tumefaciens as the vector system. Callus cell suspension cultures after transformation were assessed for significant 4-fold increase in squalene synthase activity and 2.5-fold increase in withanolide A content. However, efforts to involve tissue culture techniques in plant biotransformation may result in failure to produce metabolites in sufficient quantity as unorganized tissue cultures are unable to produce secondary metabolites at the same levels as an intact plant. Callus is a chimeric tissue, thus reducing the complete effect of the number of cells actually contributing in withanolide production. The fate of developing cells is also unknown, thus providing no idea about the specific tissue contributing for the production of secondary metabolites
There are few reports on plant cell and hairy root cultures developed for the production of the important metabolites from Withania extracts (Murthy et al. 2008; Roja et al. 1991), although withanolide production by in vitro cultures is still far from the levels required for economic exploitation. Moreover, an important constraint in the commercial utilization of hairy root culture is development and up-scaling of appropriate vessels for the delicate and sensitive hairy roots.
Agrobacterium tumefaciens mediated transformation in Withania somnifera plants is performed by Pandey et al, by employing A. tumefaciens strain LBA4404, containing binary vector p1G121Hm to obtain transgenic plants. However, absence of a functional gene in the expression vector system and minimum transformation efficiency reduces the applicability of this method.
However, these limitations can be addressed by analysing the biosynthetic pathway of withanolides (described in FIG. 1) and employing genetic engineering as a tool to manipulate crucial steps of the metabolic network to increase the yield of withanolide.
The first committed step which diverts the carbon flux away from the central isoprenoid pathway towards withanolide biosynthesis is squalene formation from farnesyl pyrophosphate (FPP); catalysed by a 47 kDa membrane associated enzyme, squalene synthase (SQS; EC 2.5.1.21) (Abe et al. 1993). The substrate for this enzyme originates from isoprenoid biosynthetic pathway and can be channelled by metabolic engineering towards squalene accumulation which is the first precursor of triterpenoids.
SQS catalyses condensation of two FPP molecules to produce presqualene diphosphate (PSPP) and then converts PSPP to squalene in presence of NADPH and Mg2+. Squalene oxidizes in presence of NADPH-linked oxide to afford squalene 2,3-epoxide subsequently cyclizing into lanosterol which serves as a backbone structure for various steroidal triterpenoids. (Mirjalili et al. 2009).
SQS being a regulatory branch point enzyme, has attracted considerable interest as a possible genetic engineering target by blocking a competing branch pathway to promote secondary metabolite biosynthesis in plants. Many approaches have been investigated to understand the regulatory role of SQS in sterol biosynthesis using SQS mutants (Karst and Lacroute, 1977; Tozawa et al., 1999), fungal elicitors (Devarenne et al., 1998; Threlfall and Whitehead, 1988; Vögeli and Chappell, 1988) and specific inhibitors of SQS (Baxter et al., 1992; Bergstrom et al., 1993; Wentzinger et al., 2002). The effect of SQS overexpression on accumulation of SMs were studied in Panax ginseng (Lee et al., 2004) and Eleutherococcus senticosus (Seo et al., 2005), and similar study was also performed in Glycyrrhiza uralensis via Ri-mediated transformation (Lu et al., 2008).
Bearing in mind the minimal concentration of withanolides in plant tissue and the disadvantages posed by chemical synthesis and tissue culture techniques to increase secondary metabolite production in Withania somnifera, the present inventors have developed a transformation process overexpressing WsSQS gene encoding squalene synthase in intact plants of W. somnifera thereby conserving the germplasm of W. somnifera, with considerable increase in withanolide content in all plant tissues.